The lectin domain of the polypeptide GalNAc transferase family of glycosyltransferases (ppGalNAc Ts) acts as a switch directing glycopeptide substrate glycosylation in an N- or C-terminal direction, further controlling mucin type O-glycosylation.

Abstract

Mucin type O-glycosylation is initiated by a large family of polypeptide GalNAc transferases (ppGalNAc Ts) that add α-GalNAc to the Ser and Thr residues of peptides. Of the 20 human isoforms, all but one are composed of two globular domains linked by a short flexible linker: a catalytic domain and a ricin-like lectin carbohydrate binding domain. Presently, the roles of the catalytic and lectin domains in peptide and glycopeptide recognition and specificity remain unclear. To systematically study the role of the lectin domain in ppGalNAc T glycopeptide substrate utilization, we have developed a series of novel random glycopeptide substrates containing a single GalNAc-O-Thr residue placed near either the N or C terminus of the glycopeptide substrate. Our results reveal that the presence and N- or C-terminal placement of the GalNAc-O-Thr can be important determinants of overall catalytic activity and specificity that differ between transferase isoforms. For example, ppGalNAc T1, T2, and T14 prefer C-terminally placed GalNAc-O-Thr, whereas ppGalNAc T3 and T6 prefer N-terminally placed GalNAc-O-Thr. Several transferase isoforms, ppGalNAc T5, T13, and T16, display equally enhanced N- or C-terminal activities relative to the nonglycosylated control peptides. This N- and/or C-terminal selectivity is presumably due to weak glycopeptide binding to the lectin domain, whose orientation relative to the catalytic domain is dynamic and isoform-dependent. Such N- or C-terminal glycopeptide selectivity provides an additional level of control or fidelity for the O-glycosylation of biologically significant sites and suggests that O-glycosylation may in some instances be exquisitely controlled.

Human ppGalNAc T phylogenetic tree and subfamilies based on Bennett et al. (). Schematics to the right represent the glycopeptide preferences obtained from this work (GPIV in red and GPV in blue), where T* represents the position of the GalNAc-O-Thr and the slanted arrow indicates the N- or C-terminal region glycosylated by the transferase. ND, transferases whose glycopeptide substrate preferences have not been determined.

A, model building of (glyco)peptide substrates GPIV and GPV onto the EA2-bound ppGalNAc T2 crystal structure (). Left, GPIV modeled with its Xaa residues placed in the catalytic domain in the same N- to C-terminal orientation as the 9 residues of the bound EA2 peptide. Right, GPV modeled in the catalytic domain. For simplicity, the 9 EA2 residues were maintained as in the original structure (see ), whereas 3 additional Ala residues were added to complete the representation of the 12-Xaa acceptor region. Likewise, Ala residues were used to represent the Zaa residues, whereas a Ser residue (green) was used to represent the location of the GalNAc-O-Thr. Note that for simplicity, both regions were modeled as static extended structures but in reality would be relatively flexible, comprising an ensemble of structures. B, superimposition of the catalytic domains of ppGalNAc T2-bound EA2 (yellow trace), ppGalNAc T2 bound UDP (blue trace), ppGalNAc T10-bound UDP and GalNAc (red trace), and ppGalNAc T1 (green trace), showing the positional variability of the lectin domain. The critical α-lectin subdomain Asp residue (see ) is space-filled. Protein Data Bank accession numbers are as follows: 2FFU (), 2FFV (), 2D7I (), and 1XHB (), respectively.

Plots of [3H]GalNAc incorporation into the Xaa residues of random glycopeptides GPIV and GPV (open bars) and after normalization by subtraction of control peptides GPIV-C or GPV-C that were glycosylated by ppGalNAc T13 (filled bars).A–H, plots of GPIV (left) and GPV (right) for ppGalNAc T1, T2, T14, T3, T6, T5, T13, and T16, respectively. Note that for A and B, substrates were glycosylated using 0.5 μm, 100-fold specific activity UDP-[3H]GalNAc, whereas in C–H, substrates were glycosylated using 50 μm standard specific activity UDP-[3H]GalNAc.